1. Volume 22, Issue 2, Pages 259-270 (February 2005)
Bone Marrow Is a Major Reservoir and Site of Recruitment for Central Memory CD8+ T Cells 
Irina B. Mazo, Marek Honczarenko, Harry Leung, Lois L. Cavanagh, Roberto Bonasio, Wolfgang Weninger, Katharina Engelke, Lijun Xia, Rodger P. McEver, Pandelakis A. Koni, Leslie E. Silberstein, Ulrich H. von Andrian 
Immunity 
Volume 22, Issue 2, Pages (February 2005)
DOI: /j.immuni
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Subset Composition of Resident CD8+ T Cells in Murine and Human BM
(A) The frequency of CD8β+ cells in BM, spleen, and blood of young adult C57BL6 mice. *p < 0.05 and **p < 0.01 versus BM (one-way ANOVA). Error bars show mean ± SEM.
(B) Activation/memory markers on CD8β+ T cells in mouse BM and spleen. Cell frequencies among all CD8β+ T cells are shown for both the naive and memory gate.
(C) CCR7 expression, as determined by staining with CCL19-Ig chimera, on naive (solid line) and memory (dotted line) CD8β+ T cells in BM. A histogram for peripheral blood memory CD8+ T cells (dashed line) is shown for comparison. Data are representative of five independent experiments.
(D) Naive and memory subset frequencies among CD8+ T cells in murine and human BM. **p < 0.01, ***p < versus TCMs, and ###p < versus naive T cells (TNs). Error bars show mean ± SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 Subset-Specific CD8+ T Cell Homing to the BM
(A) P14 TCMs and TEMs (CD45.2+) were generated in vivo and injected into CD45.1+ recipients. Frequencies of TCMs (MHC-Tet+ CCR7+) and TEMs (MHC-Tet+ CCR7−) among CD45.2+ donor cells in BM and spleen were compared with their input frequency. *p < 0.05, **p < 0.01, and ***p < versus input. #p < 0.05 and ##p < 0.01 versus corresponding subset in BM (paired Student’s t test). $p < 0.05 versus TCMs. Error bars show mean ± SEM.
(B and C) Homing of in vitro-generated TCMs, TEffs, and naive CD8+ T cells at 2 hr (B) or 24 hr (C) after adoptive transfer. A homing index (HI) of 2 means that Ag-experienced cells were twice as frequent as naive T cells, whereas HI = 1 means that both samples were equivalently represented and so on. Mean ± SEM of at least four mice are shown per group. *p < 0.05, **p < 0.01, and ***p < versus HI of TEffs (one-way ANOVA).
(D) Numbers of homed cells recovered from BM at 2 hr and 24 hr after transfer (mean ± SEM).
(E) Dose-response relationship of homed TCMs recovered from BM 2 hr after transfer of different numbers of TCMs.
(F) Representative 3D projection of optical image stacks obtained by MP-IVM showing homed TCMs in murine skull BM. Hoechst labeled TCMs (blue) were injected i.v. 3 hr prior to the recording. The intravascular compartment was delineated by i.v. injection of FITC-dextran (green, “F”). Extravascular hemopoietic tissue was stained with rhodamine 6G (red, “R”). Arrow indicates transmigrating cell. For a 3D video of this scene refer to Movie 1; for a time-lapse video showing TCMs migration in BM cavities refer to Movie 2.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3 Adhesion Pathways for TCM Homing to BM
(A) TCMs display higher intravascular adhesiveness than TEffs. Rolling fractions (RFxs), sticking fractions (SFxs), and sticking efficiencies (SEs) of TCMs and TEffs were analyzed in the same microvessels. *p < 0.05 versus TCMs. Error bars show mean ± SEM.
(B) Effect of mAbs on TCM rolling in BM venules and sinusoids. Results are shown as RFxs after mAb treatment normalized to RFxs in the same vessel prior to mAb injection (control). Error bars show mean ± SEM.
(C) TCM rolling in PSGL-1−/− mice is significantly lower than in wild-type (wt) littermates (right) but is insensitive to anti-L-selectin injection (left). Error bars show mean ± SEM.
(D) Effect of blocking mAbs to VCAM-1 and β7 integrins on TCM sticking in BM microvessels. Error bars show mean ± SEM.
(E) Sticking, but not rolling, of TCMs is compromised in VCAM-1 conditional knockout mice. Error bars show mean ± SEM.
(F) TCM homing (24 hr) in VCAM-1−/− mice. Data are presented as mean ± SEM from three to five animals. Results were compared by paired Student’s t test (A, C, E, and F) or one-way ANOVA with Bonferroni correction (B and D). *p < 0.05, **p < 0.01, and ns = not significant versus control or wt. Error bars show mean ± SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 Effect of PTX on TCM Homing and Transmigration
Recipients were sacrificed 2 hr (A) or 24 hr (B) after injection, and homing indices were calculated in indicated organ as ratio of homed PTX-treated cells to control cells (n = 4). Error bars show mean ± SEM.
(C) Representative 3D projection of MP-IVM image stacks demonstrating control (blue) and PTX-treated (red) TCM localization in skull BM 3 hr after injection. Microvessels were contrasted by FITC-dextran (green). A 3D video of this scene is shown in Movie 4.
(D) Transmigration efficiency (ratio of extra- to intravascular cells in the same field of view) of PTX treated and control TCMs. Data are from 10 to 12 fields in each of three experiment (mean ± SEM).
Results were compared by one-way ANOVA with Bonferroni correction (A and B) or paired Student’s t test (D). *p < 0.05, **p < 0.01, and ***p < versus control.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5 Role of CXCL12 in TCM Homing to BM
(A) TCMs possess higher chemotactic activity toward CXCL12 than TEffs. Inset, effect of PTX on TCM migration to 500 ng/ml CXCL12. Error bars show mean ± SEM.
(B) Effect of PTX and anti-CXCL12 on TCM homing to BM and spleen. Error bars show mean ± SEM.
(C) Effect of PTX and anti-CXCL12 on TCM sticking in BM microvessels. Error bars show mean ± SEM.
(D) Transmigration efficiency of TCMs across BM microvessels in control and anti-CXCL12-treated mice. Bars represent mean ± SEM from three to five animals. Results were compared by paired Student’s t test (A and C) or one-way ANOVA with Bonferroni correction (B). *p < 0.05, **p < 0.01, and ns = not significant.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Long-Term Adoptively Transferred TCMs in Recipient BM Respond to Recall Ag
(A) Representative dot plots showing intracellular staining of IFN-γ and IL-2 by OT-I TCMs in recipient BM 8 weeks after adoptive transfer. TCMs responded to cognate peptide (SIINFEKL), but not control gp33. Equivalent results were obtained with P14 TCMs (not shown).
(B) Frequency of Ag-specific, cytokine-producing OT-I and P14 TCMs harvested from recipient spleens and BM 8 weeks after adoptive transfer. Data were compared with a paired Student’s t test. Bars represent mean ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < versus gp33.
(C) Number of cytokine producing OT-I TCMs after stimulation with cognate peptide. *p < 0.05 versus spleen. Error bars show mean ± SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions